PROJECT SUMMARY/ABSTRACT Hematopoietic stem cells (HSCs) are arguably the best-studied adult stem cell population and are also used in the clinic in a form of bone marrow (BM) transplantation. Despite extensive efforts, the mechanism of how the BM maintains HSCs and blood homeostasis remains elusive. My laboratory has begun to explore amino acids as metabolic modulators of hematopoiesis and recently found that HSCs are highly dependent on valine. Additionally, we have found that the BM maintains high concentrations of amino acids, approximately 50-fold higher than in the peripheral blood. A valine-restricted diet can even replace irradiation to condition mice for HSC transplantation, providing important proof-of-concept for the use of dietary approaches for BM conditioning. Combined, these results led us to propose that amino acids, and in particular valine, are key components of the BM HSC microenvironment. However, several key issues remain unresolved, including how valine modulates HSC function and how amino acid concentrations are regulated within the BM. By answering these basic biological questions, we aim to improve ex vivo HSC expansion and BM transplantation protocols. In this research plan, we will: (1) define the contribution of valine catabolism to HSC function; (2) dissect the requirements of valine catabolism and amino acid uptake in the BM microenvironment; and (3) improve BM transplantation conditioning and outcome by modulating amino acid metabolism. In Aim 1, we will build on new evidence that valine catabolism regulates HSC self-renewal. While this finding helps to understand why HSCs require valine, the mechanistic relationship between HSC self-renewal and the valine catabolism pathway is not clear. To address this, we will combine metabolic approaches with in vitro and in vivo rescue assays, to define which valine catabolite(s) have ?self-renewal promoting? activity. After identifying these biologically active metabolites, we will be able to further investigate the mechanism of action and may even be able to improve ex vivo HSC expansion. Aim 2 will build on this with genetic approaches to understand the importance of valine catabolism in normal BM function. Additionally, we will test the hypothesis that BM trans-endothelial amino acid transport is responsible for maintaining high BM amino acid concentrations and sustaining hematopoiesis. In Aim 3, we will apply our understanding of HSC/BM amino acid regulation to optimize our proof-of-concept dietary BM conditioning protocols. Within the hematopoietic system, T cells and B cells are also highly dependent on valine. As these lymphoid cells are responsible for graft rejection, we will endeavor to utilize their sensitivity to valine deprivation to extend our dietary conditioning approach to allogeneic HSC transplantation. By better understanding the valine sensitivity of lymphoid subpopulations, we may even be able to develop methods to ?wipe? harmful immunological memory, such as in autoimmune type I diabetes. In summary, this project will investigate how valine acts as a modulator of hematopoiesis and how the BM regulates amino acid metabolism, with the aim of improving ex vivo HSC expansion and BM transplantation.